Electron microscopic observations on various secretory cells, frog neuromuscular junctions and adrenal medulla, have demonstrated the presence of significant numbers of vesicles covered by a proteinaceous latticework believed to be composed primarily of a single protein whose subunit molecular weight is 180,000. These vesicles are thought to be involved in recycling of fused membrane subsequent to secretory product release. The properties of this protein, clathrin, currently are little understood, although it has been identified in brain, adrenal and oocyte-coated vesicles. The intent of this proposal is to focus on the in vitro biochemical and biophysical properties of clathrin in order to provide answers to some of the outstanding questions relating to its role in secretory activity. Special emphasis is placed on studies designed to determine how clathrin associates to form the basket-like assembly characteristic of coated vesicles. Biochemical and biophysical analysis of its interaction with the various filamentous proteins (actin, tubulin, alpha-actinin) known to be present in secretory cells will be carried out. To localize and further describe the interactions of these proteins in vivo, immunological labeling techniques with antibodies purified by affinity chromatography and conjugated with markers will be carried out. With the studies proposed, we attempt to explain the mechanics by which certain cells transport their vesicles' packaged metabolic products to their exteriors and reutilize useful membrane fragments.